Appropriate conditioning process is required prior to a steady-state operation of direct methanol fuel cell (DMFC). Different methods for conditioning a membrane-electrode assembly (MEA) were applied, and the conditioning periods were generally between 12 and 48 hours except in a special case. The conditioning process affected the cell performance by some variables such as conditioning temperature, circulation of reactants, and application of electric load. In this study, we identified the major factor for it and elucidated the phenomena occurring in the anode during conditioning. In order to measure the resistance and the capacitance affecting the performance of MEAs, Nyquist plots were obtained by using an impedance analysis technique.